The effect of copper(II) ions (Cu+2) on the structure of β-lactoglobulin (β-lg) was investigated spectroscopically using UV-visible, fluorescence and circular dichroism (CD) and calorimetrically using isothermal titration calorimetry (ITC), at different temperatures. Results of the UV-visible studies showed that adding Cu+2 to β-lg solution caused increasing turbidity, indicative of protein aggregation. It was noticeable that the rate of increasing turbidity was directly proportional to increasing temperature. The far-UV CD studies displayed that the Cu+2 cannot induce any significant changes in the secondary structures of β-lg at different temperatures. Also, the ITC data indicated that the binding process of Cu+2 to β-lg is mainly entropically driven. The results highlight that copper ions cause the tertiary structure of β-lg to change and induce a slightly open structure leading to the formation of supramolecular aggregates in β-lg which may result in the reduced allergenicity of β-lg and its increased use in industrial applications.

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 °C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 °C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.